Process for preparing a protein which inhibits metastasis of cancer

ABSTRACT

Disclosed is a novel protein which has a molecular weight of 45,000±5,000 and pI 5.7±0.5 and exhibits cancer metastasis-inhibitory activity. The protein can be prepared by culturing human cells, animal cells and microorganisms capable of producing the protein in a nutrient culture medium while stimulating them with an inducer such as Bacille Calmette-Guerin and lipopolysaccharide.

This application is a continuation, of application Ser. No. 08/555,857,filed Nov. 13, 1995, now abandoned is a division of parent applicationSer. No. 08/127,278 filed Sep. 27, 1993, now U.S. Pat. No. 5,498,697.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel protein, and a DNA coding saidprotein, as well as to the preparation of said protein.

2. Description of the Prior Art

Nowadays, the treatment of cancers is mainly by surgical operations,chemotherapies and radiotherapies. Although most cancers may be cured bysuch a treatment, some viable cancer cells remaining after such atreatment may be, scattered throughout the body of a cancer patient, andmay cause a more serious cancer-metastasis and even shorten thepatient's life span. If the metastasis of cancers can be inhibited,cancer patients's pain would be relieved, and their life spans would beextended much more. Therefore, the development of agents whicheffectively inhibit the metastasis of cancers has been in great demand.In general, the metastasis of cancers, however, has been considered tooccur via a complicated process, and this hinders the realization ofsatisfiable cancer metastasis-inhibitory agents.

Although a variety of proteins possessing cancer metastasis-inhibitoryactivity have been reported, the present protein absolutely differs fromthem. Examples of such a conventional protein are interferons andinterleukin 2 which have been reported to have cancermetastasis-inhibitory activity. The present protein clearly differs fromsuch a conventional protein in molecular weight and amino acid sequence.No conventional proteins have yet been realized as a cancermetastasis-inhibitory agent. In Japanese Patent Laid-Open No.308,799/90,a cancer metastasis-inhibitory factor produced by cells derived fromhuman hematopoietic tissues is reported and its structure andphysicochemical properties are, however, far from substantialelucidation because the description in the patent is vague and it onlyteaches the molecular weight ranging 10,000-450,000.

OBJECTS OF THE INVENTION

One object of the present invention is to provide a novel protein whicheffectively inhibits the metastasis of cancers.

Another object of the invention is to provide a DNA coding said protein.

Further object of the invention is to provide the preparation of saidprotein.

SUMMARY OF THE INVENTION

In order to attain the aforesaid objects, the present inventors studiedsubstances which inhibit the metastasis of cancers. The presentinventors continued studies in order to obtain a novel cancermetastasis-inhibitory substance, and, as a result eventually foundcancer metastasis-inhibitory activity in a culture supernatant ofHPB-MLT cell (FERM BP-2430), an established cell line derived from humanT-cell leukemia, which had been stimulated in a nutrient culture mediumwith BCG and LPS. The present inventors revealed that the entity of theactivity is a protein having the following physicochemical properties:

(1) Molecular weight 45,000±5,000;

(2) Isoelectric point pI=5.7±0.5;

(3) Partial amino acid sequence Possessing a partial amino acid sequenceof Asp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys (SEQ IDNO:1) or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu (SEQ ID NO: 2);

(4) Solubility in solvent Soluble in water, physiological saline andphosphate buffer;

(5) Biological activity Exhibiting a metastasis-inhibitory activity onRPMI 4788 cell (FERM BP-2429), an established cell line derived fromhuman colon cancer; and

(6) Stability Inactivated in water at pH 7.2 and 100° C. for 30 minutes.

Stable in water at pH 7.2 and 4° C. for one month.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors isolated a protein possessing cancermetastasis-inhibitory activity from a culture of HPB-MLT cell (FERMBP-2430) stimulated with BCG and LPS. The present inventors revealed thephysicochemical properties of said protein and found that it has theamino acid sequence as shown in Chemical formula 1 (SEQ ID NO:4). Thewording "substantially has the amino acid sequence as shown in Chemicalformula 1" as referred to in the invention means that it should not berestricted to that as shown in Chemical formula 1 and shall include allthe homologous variants thereof. In other words, the present inventionincludes any protein having a partial amino acid sequence in the aminoacid sequence as shown in Chemical formula 1 as long as it possesses thesame physicochemical properties as the above-mentioned protein.

    ______________________________________                                        Chemical formula 1 (SEQ ID no:4)                                              ______________________________________                                        1      Met--Thr--Ser--Lys--Gly--Pro--Glu--Glu--Glu--His--                     11     Pro--Ser--Val--Thr--Leu--Phe--Arg--Gln--Tyr--Leu--                     21     Arg--Ile--Arg--Thr--Val--Gln--Pro--Lys--Pro--Asp--                     31     Tyr--Gly--Ala--Ala--Val--Ala--Phe--Phe--Glu--Glu--                     41     Thr--Ala--Arg--Gln--Leu--Gly--Leu--Gly--Cys--Gln--                     51     Lys--Val--Glu--Val--Ala--Pro--Gly--Tyr--Val--Val--                     61     Thr--Val--Leu--Thr--Trp--Pro--Gly--Thr--Asn--Pro--                     71     Thr--Leu--Ser--Ser--Ile--Leu--Leu--Asn--Ser--His--                     81     Thr--Asp--Val--Val--Pro--Val--Phe--Lys--Glu--His--                     91     Trp--Ser--His--Asp--Pro--Phe--Glu--Ala--Phe--Lys--                     101    Asp--Ser--Glu--Gly--Tyr--Ile--Tyr--Ala--Arg--Gly--                     111    Ala--Gln--Asp--Met--Lys--Cys--Val--Ser--Ile--Gln--                     121    Tyr--Leu--Glu--Ala--Val--Arg--Arg--Leu--Lys--Val--                     131    Glu--Gly--His--Arg--Phe--Pro--Arg--Thr--Ile--His--                     141    Met--Thr--Phe--Val--Pro--Asp--Glu--Glu--Val--Gly--                     151    Gly--His--Gln--Gly--Met--Glu--Leu--Phe--Val--Gln--                     161    Arg--Pro--Glu--Phe--His--Ala--Leu--Arg--Ala--Gly--                     171    Phe--Ala--Leu--Asp--Glu--Gly--Ile--Ala--Asn--Pro--                     181    Thr--Asp--Ala--Phe--Thr--Val--Phe--Tyr--Ser--Glu--                     191    Arg--Ser--Pro--Trp--Trp--Val--Arg--Val--Thr--Ser--                     201    Thr--Gly--Arg--Pro--Gly--His--Ala--Ser--Arg--Phe--                     211    Met--Glu--Asp--Thr--Ala--Ala--Glu--Lys--Leu--His--                     221    Lys--Val--Val--Asn--Ser--Ile--Leu--Ala--Phe--Arg--                     231    Glu--Lys--Glu--Trp--Gln--Arg--Leu--Gln--Ser--Asn--                     241    Pro--His--Leu--Lys--Glu--Gly--Ser--Val--Thr--Ser--                     251    Val--Asn--Leu--Thr--Lys--Leu--Glu--Gly--Gly--Val--                     261    Ala--Tyr--Asn--Val--Ile--Pro--Ala--Thr--Met--Ser--                     271    Ala--Ser--Phe--Asp--Phe--Arg--Val--Ala--Pro--Asp--                     281    Val--Asp--Phe--Lys--Ala--Phe--Glu--Glu--Gln--Leu--                     291    Gln--Ser--Trp--Cys--Gln--Ala--Ala--Gly--Glu--Gly--                     301    Val--Thr--Leu--Glu--Phe--Ala--Gln--Lys--Trp--Met--                     311    His--Pro--Gln--Val--Thr--Pro--Thr--Asp--Asp--Ser--                     321    Asn--Pro--Trp--Trp--Ala--Ala--Phe--Ser--Arg--Val--                     331    Cys--Lys--Asp--Met--Asn--Leu--Thr--Leu--Glu--Pro--                     341    Glu--Ile--Met--Pro--Ala--Ala--Thr--Asp--Asn--Arg--                     351    Tyr--Ile--Arg--Ala--Val--Gly--Val--Pro--Ala--Leu--                     361    Gly--Phe--Ser--Pro--Met--Asn--Arg--Thr--Pro--Val--                     371    Leu--Leu--His--Asp--His--Asp--Glu--Arg--Leu--His--                     381    Glu--Ala--Val--Phe--Leu--Arg--Gly--Val--Asp--Ile--                     391    Tyr--Thr--Arg--Leu--Leu--Pro--Ala--Leu--Ala--Ser--                     401    Val--Pro--Ala--Leu--Pro--Ser--Asp--Ser                                 ______________________________________                                    

Based on the above-mentioned amino acid sequence, the present inventorsscreened DNAs which might code the present protein from HPB-MLT cell,and found that the present protein contained the base sequence as shownin Chemical formula 2 (SEQ ID NO:3). The DNA according to the presentinvention is not restricted to that as shown in Chemical formula 2. Thewording "substantially has the base sequence as shown in Chemicalformula 2" as referred to in the invention means that it has the wholeor a part of the base sequence of Chemical formula 2. The base sequencesusable in the invention are, for example, those formed by a genetic codedegeneracy wherein one or more bases in Chemical formula 2 are replacedwith other bases, those which code the aforesaid homologous variants,and those which are complementary to the base sequence as shown inChemical formula 2. The complementary base sequences may be wholly orpartially complementary to that as shown in Chemical formula 2.

    __________________________________________________________________________    Chemical formula 2 (SEQ ID no:3)                                              __________________________________________________________________________    1  ATG ACC                                                                              AGC AAG                                                                              GGT CCC                                                                              GAG GAG                                                                              GAG CAC                                        31 CCA TCG                                                                              GTG ACG                                                                              CTC TTC                                                                              CGC CAG                                                                              TAC CTG                                        61 CGT ATC                                                                              CGC ACT                                                                              GTC CAG                                                                              CCC AAG                                                                              CCT GAC                                        91 TAT GGA                                                                              GCT GCT                                                                              GTG GCT                                                                              TTC TTT                                                                              GAG GAG                                        121                                                                              ACA GCC                                                                              CGC CAG                                                                              CTG GGC                                                                              CTG GGC                                                                              TGT CAG                                        151                                                                              AAA GTA                                                                              GAG GTG                                                                              GCA CCT                                                                              GGC TAT                                                                              GTG GTG                                        181                                                                              ACC GTG                                                                              TTG ACC                                                                              TGG CCA                                                                              GGC ACC                                                                              AAC CCT                                        211                                                                              ACA CTC                                                                              TCC TCC                                                                              ATC TTG                                                                              CTC AAC                                                                              TCC CAC                                        241                                                                              ACG GAT                                                                              GTG GTG                                                                              CCT GTC                                                                              TTC AAG                                                                              GAA CAT                                        271                                                                              TGG AGT                                                                              CAC GAC                                                                              CCC TTT                                                                              GAG GCC                                                                              TTC AAG                                        301                                                                              GAT TCT                                                                              GAG GGC                                                                              TAC ATC                                                                              TAT GCC                                                                              AGG GGT                                        331                                                                              GCC CAG                                                                              GAC ATG                                                                              AAG TGC                                                                              GTC AGC                                                                              ATC CAG                                        361                                                                              TAC CTG                                                                              GAA GCT                                                                              GTG AGG                                                                              AGG CTG                                                                              AAG GTG                                        391                                                                              GAG GGC                                                                              CAC CGG                                                                              TTC CCC                                                                              AGA ACC                                                                              ATC CAC                                        421                                                                              ATG ACC                                                                              TTT GTG                                                                              CCT GAT                                                                              GAG GAG                                                                              GTT GGG                                        451                                                                              GGT CAC                                                                              CAA GGC                                                                              ATG GAG                                                                              CTG TTC                                                                              GTG CAG                                        481                                                                              CGG CCT                                                                              GAG TTC                                                                              CAC GCC                                                                              CTG AGG                                                                              GCA GGC                                        511                                                                              TTT GCC                                                                              CTG GAT                                                                              GAG GGC                                                                              ATA GCC                                                                              AAT CCC                                        541                                                                              ACT GAT                                                                              GCC TTC                                                                              ACT GTC                                                                              TTT TAT                                                                              AGT GAG                                        571                                                                              CGG AGT                                                                              CCC TGG                                                                              TGG GTG                                                                              CGG GTT                                                                              ACC AGC                                        601                                                                              ACT GGG                                                                              AGG CCA                                                                              GGC CAT                                                                              GCC TCA                                                                              CGC TTC                                        631                                                                              ATG GAG                                                                              GAC ACA                                                                              GCA GCA                                                                              GAG AAG                                                                              CTG CAC                                        661                                                                              AAG GTT                                                                              GTA AAC                                                                              TCC ATC                                                                              CTG GCA                                                                              TTC CGG                                        691                                                                              GAG AAG                                                                              GAA TGG                                                                              CAG AGG                                                                              CTG CAG                                                                              TCA AAC                                        721                                                                              CCC CAC                                                                              CTG AAA                                                                              GAG GGG                                                                              TCC GTG                                                                              ACC TCC                                        751                                                                              GTG AAC                                                                              CTG ACT                                                                              AAG CTA                                                                              GAG GGT                                                                              GGC GTG                                        781                                                                              GCC TAT                                                                              AAC GTG                                                                              ATA CCT                                                                              GCC ACC                                                                              ATG AGC                                        811                                                                              GCC AGC                                                                              TTT GAC                                                                              TTC CGT                                                                              GTG GCA                                                                              CCG GAT                                        841                                                                              GTG GAC                                                                              TTC AAG                                                                              GCT TTT                                                                              GAG GAG                                                                              CAG CTG                                        871                                                                              CAG AGC                                                                              TGG TGC                                                                              CAG GCA                                                                              GCT GGC                                                                              GAG GGG                                        901                                                                              GTC ACC                                                                              CTA GAG                                                                              TTT GCT                                                                              CAG AAG                                                                              TGG ATG                                        931                                                                              CAC CCC                                                                              CAA GTG                                                                              ACA CCT                                                                              ACT GAT                                                                              GAC TCA                                        961                                                                              AAC CCT                                                                              TGG TGG                                                                              GCA GCT                                                                              TTT AGC                                                                              CGG GTC                                        991                                                                              TGC AAG                                                                              GAT ATG                                                                              AAC CTC                                                                              ACT CTG                                                                              GAG CCT                                        1021                                                                             GAG ATC                                                                              ATG CCT                                                                              GCT GCC                                                                              ACT GAC                                                                              AAC CGC                                        1051                                                                             TAT ATC                                                                              CGC GCG                                                                              GTG GGG                                                                              GTC CCA                                                                              GCT CTA                                        1081                                                                             GGC TTC                                                                              TCA CCC                                                                              ATG AAC                                                                              CGC ACA                                                                              CCT GTG                                        1111                                                                             CTG CTG                                                                              CAC GAC                                                                              CAC GAT                                                                              GAA CGG                                                                              CTG CAT                                        1141                                                                             GAG GCT                                                                              GTG TTC                                                                              CTC CGT                                                                              GGG GTG                                                                              GAC ATA                                        1171                                                                             TAT ACA                                                                              CGC CTG                                                                              CTG CCT                                                                              GCC CTT                                                                              GCC AGT                                        1201                                                                             GTG CCT                                                                              GCC CTG                                                                              CCC AGT                                                                              GAC AGC                                               __________________________________________________________________________

Furthermore, the present invention provides a process to prepare theabove-mentioned protein, comprising culturing a cell capable ofproducing said protein in a nutrient culture medium to form saidprotein, and recovering the resultant protein from the culture. Examplesof such a cell are established cell lines derived from human T-cellleukemia such as HPB-MLT cell and MOLT-4 cell (ATCC CRL 1582), and notrestricted to those of human origin. Similarly as the cells of humanorigin, cells of animal origin and microorganisms can be advantageouslyused in the invention as long as they can inherently produce the presentprotein, and those into which had been introduced the present DNA byconventional cell fusion or genetic engineering technique can beadvantageously used in the invention as long as the present protein isrecovered from their cultures.

The cells usable in the present process are not restricted to thosedescribed in the present specification, and, if necessary the presentprotein can be prepared by culturing any one of the cells in a nutrientculture medium while stimulating it with an adequate stimulant such asBCC and LPS, and recovering the resultant protein having ametastasis-inhibitory activity from the resultant cells or supernatant.The cultivation of such a cell is carried out according to conventionaltechniques for animal cells and microorganisms. Conventional nutrientculture media containing vitamins, minerals, carbohydrates and the likecan be employed in the invention. The recovering methods suitably usedin the invention are two or more methods usually used in thepurification of physiologically-active proteinous substances: Forexample, salting out, dialysis, centrifugation, gel filtrationchromatography, ion-exchange chromatography, affinity chromatography,electrophoresis, isoelectrofocusing and isoelectric fractionation aresuitably used in combination.

The present invention attains the aforesaid object, and encompasses anovel protein possessing a metastasis-inhibitory activity, a DNA codingsaid protein, and the preparation of said protein.

The following experiments will explain the present invention.

EXPERIMENT 1

Assay for metastasis-inhibitory activity

In accordance with the method of Y. Naomoto et al. described in Journalof Cancer Research and Clinical Oncology, Vol.113, pp.544-549 (1987), ametastasis-inhibitory activity was assayed with a model wherein RPMI4788 cells (FERM BP-2429) are transplanted to nude mice so as to inducelung metastasis.

In a test group, 5 or more BALB/c nude mice were injected through theirtail veins with 0.2 ml of phosphate buffer containing a test specimen 3times in total before the cell transplantation, i.e. at 2 day, 1 day and3 hours before the cell transplantation. From the next day of thetransplantation of 2×10⁶ RPMI 4788 cells per mouse, the mice weresuccessively injected similarly as above with a test specimen one shotper day over a period of 7 days. In a control group, mice were similarlytreated as in the test group except for using phosphate buffer free ofthe test specimen. On the 21st day after the cell transplantation, nudemice were sacrificed, and the number of metastatic nodules formed on thesurfaces of the lungs was macroscopically counted. It was judged that atest specimen had a positive activity when the following requirementswere fulfilled: (i) The mean value of numbers of lung metastatic nodulesformed in the mice in the control group was 50 or more; (ii) the meanvalue of those in the test group was lowered to 1/2 or lower againstthat in the control group; and (iii) the reduction level in (ii) wasevaluated as statistically significant.

EXPERIMENT 2

Preparation of supernatant from culture of HPB-MLT cell stimulated withBCG and LPS

A new born hamster was first injected with a serum of anti-hamsterthymus prepared from rabbits in usual manner, then subcutaneouslytransplanted with HPB-MLT cells, and bred for 4 weeks in usual manner.About 20 g weight tumor subcutaneously formed in the hamster was cutinto pieces, dispersed, washed with serum-free RPMI 1640 medium, andsuspended in a fresh preparation of the same medium to give aconcentration of 5×10⁶ cells/ml. The cell suspension was added with 10μg/ml BCG and incubated at 37° C. for one day. Thereafter, the resultantcell suspension was added with one μg/ml LPS, incubated for one day, andcentrifuged to obtain a supernatant.

EXPERIMENT 3

Purification and physicochemical properties of the present protein

The supernatant in Experiment 2 was concentrated by about 20-fold on"AIL 3013", a membrane module commercialized by Asahi Chemical Ind.,Tokyo, Japan, and the concentrate was dialyzed against 25 mMimidazol-HCl buffer (pH 7.4). The resultant solution in a dialytic bagwas fed to a column packed with "PBE-94", a product of Pharmacia LKB,Uppsala, Sweden, preequilibrated with 25 mM imidazol-HCl buffer (pH7.4). The column was fed with "Polybuffer 74 (pH 4.0)", commercializedby Pharmacia LKB, Uppsala, Sweden, to fractionate the supernatant, andthe resultant fractions were respectively dialyzed againstphosphate-buffered saline (PBS), followed by assaying each fraction forcancer metastasis-inhibitory activity. As a result, it was revealed thatthe fractions eluted between a pH range of 5.0-6.5 had cancermetastasis-inhibitory activity. The active fractions were pooled, andrefractionated on a column packed with "Sephacryl S-200", a productcommercialized by Pharmacia LKB Uppsala, Sweden. The resultant fractionswere assayed for their cancer metastasis-inhibitory activity to testwhether or not that the fractions, eluted with a ratio of (Elutionvolume)/(Void volume) in the range of 0.5-0.65, might have the activity.The active fractions were pooled and dialyzed against 10 mM potassiumphosphate buffer (pH 7.4). The solution in a dialytic bag was fed to acolumn packed with "DEAE-5PW", a product of Tosoh Corporation, Tokyo,Japan, preequilibrated with 10 mM potassium phosphate buffer (pH 7.4),and eluted with a liner gradient of 10-500 mM potassium phosphate buffer(pH 7.4). In this case, a substance with cancer metastasis-inhibitoryactivity was eluted in fractions with about 70 mM potassium phosphate.The fractions thus obtained were pooled and dialyzed against 10 mMsodium phosphate buffer (pH 6.8), and fed to a hydroxyapatite columncommercialized by Toa Nenryo Kogyo K.K., Tokyo, Japan, preequilibratedwith 10 mM sodium phosphate buffer (pH 6.8). In this case, cancermetastasis-inhibitory activity was found in non-adsorbed fractions whichwere then fed to a column packed with "Mono-P", a product of PharmaciaLKB, Upssala, Sweden, preequilibrated with 25 mM bis-tris-iminodiacetatebuffer (pH 7.1), and eluted with "Polybuffer® 74 (pH 4.0)", a product ofPharmacia LKB, Uppsala, Sweden, followed by isolating the presentprotein with cancer metastasis-inhibitory activity. Thus about 70 μg ofthe present protein was isolated from 50L of the culture supernatant ofHPB-MLT cells stimulated with BCG and LPS. The physicochemicalproperties of the present protein were studied with the isolatedprotein.

(1) Molecular weight In accordance with the method of U. K. Laemmlidescribed in Nature, Vol.227, pp.680-685 (1970), the protein wassubjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis(abbreviated as "SDS-PAGE" hereinafter), and the molecular weight wasdetermined to be 45,000±5,000 based on the relative mobility of theprotein against marker proteins.

(2) Isoelectric point The isoelectric point of the protein was estimatedto be 5.7±0.5 based on the pHs of eluates on a column chromatographyusing Mono-P column;

(3) Partial amino acid sequence The protein was subjected to SDS-PAGE,and a band corresponding to the molecular weight of about 45,000 in theresultant gel was isolated by cutting. The resultant gel piece wassoaked in 100 mM Tris-HCl buffer (pH 9.0) containing 0.1% sodium dodecylsulfate (SDS) at 37° C. for one hour, and digested at 37° C. overnightby the addition of 5 μg/ml lysyl endopeptidase, an enzyme specimencommercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan. Thesupernatant obtained from the resultant was fractionated on areverse-phase chromatography using a column of "C-18", commercialized byVYDAC, Hesperia, USA, equipped with a precolumn of DEAE, followed byrecovering peptide fragments which were then analyzed on "470A", anamino acid sequencer commercialized by Applied Biosystems Inc., FosterCity, USA. The results were as shown in Chemical formulas 3, 4, 5 and 6.##STR1## (4) Solubility in solvent The protein was soluble in water,physiological saline and phosphate buffer.

(5) Biological activity The protein was tested for cancermetastasis-inhibitory activity with the method in Experiment 1. As aresult, the number of metastatic nodules was 314+169 in a control groupconsisting of 5 nude mice, while that in a test group, wherein 5 nudemice were administered with a solution containing 250 μg/ml of theprotein, was 100±43.

These confirmed that the protein exhibited a strong cancermetastasis-inhibitory activity.

(6) Stability The protein was dissolved in phosphate buffer (pH 7.2) andallowed to stand at 100° C. for 30 minutes, followed by determining theresidual activity with the method in Experiment 1 to give no activity.Thus, it was revealed that the protein was inactivated under theconditions. While the protein was treated by dissolving it in phosphatebuffer (pH 7.2), and allowing the resultant solution to stand at 4° C.for one month, followed by assaying the residual activity similarly asabove. As a result, no substantial loss of activity was found, and thisrevealed that the protein was stable under the conditions.

EXPERIMENT 4

Acute toxicity

By using 7 week-old mice, the present protein was tested for acutetoxicity. As a result, the LD₅₀ in mice of the protein was 50 mg/kg orhigher when intravenously administered to the mice, and this revealedthat the toxicity was extremely low.

EXPERIMENT 5

Base sequence coding the present protein

In this experiment, the base sequence of the present protein wasdetermined by conventional method as described by T. Maniatis et al. inMolecular Cloning, A Laboratory Manual, 2nd edition, published by ColdSpring Harbor Laboratory Press (1989), New York, USA.

EXPERIMENT 5-1

Construction of cDNA library of HPB-MLT cell

HPB-MLT cells obtained by the method in Experiment 2 were suspended in aserum-free RPMI 1640 medium to give a concentration of 5×10⁶ cells/ml.The cell suspension was added with 10 μg/ml BCG and incubated at 37° C.for one day. The culture thus obtained was added with one μg/ml LPS andincubated for 4.5 hours. The resultant culture was centrifuged to obtaincells which were then solubilized with 4M guanidium isocyanate andhomogenized. The resultant homogenate was overlaid on 5.7M cesiumchloride, and the mixture was centrifuged at 25,000 rpm for 17 hours toobtain the whole RNAs of the cells. Poly (A)+RNA was purified from thewhole RNAs on "Oligotex®-dT30<Super>", a bead for purification of poly(A)+RNA commercialized by Daiichi Pure Chemicals, Tokyo, Japan. Thepurified poly (A)⁺ RNA was treated with "cDNA synthesis system plus", aproduct of Amersham International plc, Buckinghashire, England, tosynthesize a cDNA under the direction of the appended manual. The cDNAthus obtained was ligated with a λgt10 phage DNA by using "cDNA cloningsystem λgt10", a product of Amersham International plc, Buckinghamshire,England. The resultant recombinant phage DNA was packaged by "Lambda(λ)in vitro packaging kit", a product of Amersham International plc,Buckinghamshire, England, to obtain a cDNA library of HPB-MLT cell.

EXPERIMENT 5-2

Construction of radiolabeled DNA probe

Based on the partial amino acid sequence as shown in Chemical formula 3in Experiment 3, base sequences estimable from the amino acid sequenceof Glu-Gly-Tyr-Ile-Tyr-Ala (SEQ ID NO:7) in Chemical formula 3 weresynthesized by a DNA synthesizer commercialized by Applied Biosystems,Inc., Foster City, USA. Ninety-six base sequences consisting of 17synthesized bases are as shown in Table 1.

                  TABLE 1                                                         ______________________________________                                         ##STR2##                                                                     ______________________________________                                    

As for the partial amino acid sequence as shown in Chemical formula 5,complementary chains of base sequences, estimable fromAsn-Pro-His-Leu-Lys (SEQ ID NO:9) in the partial amino acid sequence inChemical formula 5, were synthesized similarly as above. Ninety-six basesequences consisting of 14 synthesized bases are as shown in Table 2.The DNAs thus obtained were radiolabeled with γ-³² p! ATP and T4polynucleotide kinase.

                  TABLE 2                                                         ______________________________________                                         ##STR3##                                                                     ______________________________________                                    

EXPERIMENT 5-3

Screening with radiolabeled DNA probes

A solution of the recombinant λgt10, a cDNA library of HPB-MLT cellprepared in Experiment 5-1, was mixed with an overnight culture ofmicroorganisms of E. coli strain NM 514 in L-broth, and the mixture wasincubated at 37° C. for 15 minutes. The resultant was admixed with asoft agar, and the resultant mixture was overlaid on a hard-agar plateand solidified. The resultant agar plate containing the microorganismswere incubated at 37° C. for 8 hours, cooled and overlaid with "Hybond-Nfilter", a membrane filter of Amersham International plc,Buckinghamshire, England, to transfer the phage and to fix the phage DNAon the membrane filter. In order to prevent a non-specific bonding ofthe radiolabeled DNA probes with DNAs except for the objectivecomplementary DNA, the membrane filter was soaked in a solution of asalmon sperm DNA commercialized by Sigma Chemical company, St., Louis,USA, to effect prehybridization, followed by screening positive clonesby the southern hybridization with the radiolabeled DNA probes inExperiment 5-2. The results in the first screening test with the probe 1in Table 1 and the second screening test with the probe 2 in Table 2revealed that 3 positive clones were present among about 600,000 clones.Phage DNAs isolated from the positive clones were digested with arestriction enzyme EcoRI to remove them from vector DNAs, and the lengthof the inserted fragments were studied on agarose electrophoresis,followed by analyzing a clone having the longest inserted-fragment ofabout 1.5 kbp.

EXPERIMENT 5-4

Base sequence of gene coding the present protein

From the positive clones obtained in Experiment 5-3, a phage DNA wasisolated and digested with a restriction enzyme EcoRI, and the resultantfragments were separated on SDS-PAGE to obtain an inserted DNA fragmentof about 1.5 kbp which was then ligated with a pUC18 plasmid with aligation kit commercialized by Amersham International plc,Buckinghashire, England, to obtain a recombinant plasmid. Therecombinant plasmid thus obtained was introduced in usual manner into E.coli to obtain a recombinant microorganism, and from which a plasmid DNAwas prepared. The dideoxy chain termination method was applied to theresultant plasmid DNA to reveal the base sequence of the presentprotein. Based on the base sequence, the amino acid sequence of thepresent protein was estimated. As a result, it was revealed that thepresent DNA consisted of 1,224 bases and coded a protein consisting of408 amino acids. The estimated amino acid sequence encompassed the aminoacid sequences as shown in Chemical formulas 3, 4, 5 and 6 in Experiment3.

The following is an example of the preparation of the present protein.

EXAMPLE

Similarly as the method in Experiment 2, MOLT-4 cell (ATCC CRL 1582), anestablished cell line derived from human T-cell leukemia commercializedby Dainippon Pharmaceutical Co., Ltd., Tokyo, Japan, were cultured in anutrient culture medium while stimulating the cells with BCG and LPS.Similarly as in Experiment 3, a supernatant prepared from the resultantculture was treated to obtain the present protein possessing cancermetastasis-inhibitory activity. The yield was about 40 μg per 50L of theculture supernatant. The protein had the same physicochemical propertiesas the one in Experiment 3.

The protein according to the present invention effectively inhibits themetastasis of cancers, and this renders it advantageously useful asprophylactic-, therapeutic- and diagnostic-agents for cancer metastases.The toxicity of the present protein is satisfactorily low, because ofthis it can be systematically administered to patients in the form of aninjection, sublingual agent, and the like.

The present protein having the aforesaid advantages is prepared in anindustrial scale by the present preparation.

The DNA coding the present protein is useful in the preparation of thepresent protein by genetic engineering techniques.

While there has been described what is at present considered to be thepreferred embodiments of the invention, it will be understood thevarious modifications may be made therein, and it is intended to coverthe appended claims all such modifications as fall within the truespirits and scope of the invention.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 11                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspSerGluGlyTyrIleTyrAlaArgGlyAlaGlnAspMetLys                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GluHisTrpSerHisAspProPheGlu                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1224 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1224                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGACCAGCAAGGGTCCCGAGGAGGAGCACCCATCGGTGACGCTCTTC48                            MetThrSerLysGlyProGluGluGluHisProSerValThrLeuPhe                              151015                                                                        CGCCAGTACCTGCGTATCCGCACTGTCCAGCCCAAGCCTGACTATGGA96                            ArgGlnTyrLeuArgIleArgThrValGlnProLysProAspTyrGly                              202530                                                                        GCTGCTGTGGCTTTCTTTGAGGAGACAGCCCGCCAGCTGGGCCTGGGC144                           AlaAlaValAlaPhePheGluGluThrAlaArgGlnLeuGlyLeuGly                              354045                                                                        TGTCAGAAAGTAGAGGTGGCACCTGGCTATGTGGTGACCGTGTTGACC192                           CysGlnLysValGluValAlaProGlyTyrValValThrValLeuThr                              505560                                                                        TGGCCAGGCACCAACCCTACACTCTCCTCCATCTTGCTCAACTCCCAC240                           TrpProGlyThrAsnProThrLeuSerSerIleLeuLeuAsnSerHis                              65707580                                                                      ACGGATGTGGTGCCTGTCTTCAAGGAACATTGGAGTCACGACCCCTTT288                           ThrAspValValProValPheLysGluHisTrpSerHisAspProPhe                              859095                                                                        GAGGCCTTCAAGGATTCTGAGGGCTACATCTATGCCAGGGGTGCCCAG336                           GluAlaPheLysAspSerGluGlyTyrIleTyrAlaArgGlyAlaGln                              100105110                                                                     GACATGAAGTGCGTCAGCATCCAGTACCTGGAAGCTGTGAGGAGGCTG384                           AspMetLysCysValSerIleGlnTyrLeuGluAlaValArgArgLeu                              115120125                                                                     AAGGTGGAGGGCCACCGGTTCCCCAGAACCATCCACATGACCTTTGTG432                           LysValGluGlyHisArgPheProArgThrIleHisMetThrPheVal                              130135140                                                                     CCTGATGAGGAGGTTGGGGGTCACCAAGGCATGGAGCTGTTCGTGCAG480                           ProAspGluGluValGlyGlyHisGlnGlyMetGluLeuPheValGln                              145150155160                                                                  CGGCCTGAGTTCCACGCCCTGAGGGCAGGCTTTGCCCTGGATGAGGGC528                           ArgProGluPheHisAlaLeuArgAlaGlyPheAlaLeuAspGluGly                              165170175                                                                     ATAGCCAATCCCACTGATGCCTTCACTGTCTTTTATAGTGAGCGGAGT576                           IleAlaAsnProThrAspAlaPheThrValPheTyrSerGluArgSer                              180185190                                                                     CCCTGGTGGGTGCGGGTTACCAGCACTGGGAGGCCAGGCCATGCCTCA624                           ProTrpTrpValArgValThrSerThrGlyArgProGlyHisAlaSer                              195200205                                                                     CGCTTCATGGAGGACACAGCAGCAGAGAAGCTGCACAAGGTTGTAAAC672                           ArgPheMetGluAspThrAlaAlaGluLysLeuHisLysValValAsn                              210215220                                                                     TCCATCCTGGCATTCCGGGAGAAGGAATGGCAGAGGCTGCAGTCAAAC720                           SerIleLeuAlaPheArgGluLysGluTrpGlnArgLeuGlnSerAsn                              225230235240                                                                  CCCCACCTGAAAGAGGGGTCCGTGACCTCCGTGAACCTGACTAAGCTA768                           ProHisLeuLysGluGlySerValThrSerValAsnLeuThrLysLeu                              245250255                                                                     GAGGGTGGCGTGGCCTATAACGTGATACCTGCCACCATGAGCGCCAGC816                           GluGlyGlyValAlaTyrAsnValIleProAlaThrMetSerAlaSer                              260265270                                                                     TTTGACTTCCGTGTGGCACCGGATGTGGACTTCAAGGCTTTTGAGGAG864                           PheAspPheArgValAlaProAspValAspPheLysAlaPheGluGlu                              275280285                                                                     CAGCTGCAGAGCTGGTGCCAGGCAGCTGGCGAGGGGGTCACCCTAGAG912                           GlnLeuGlnSerTrpCysGlnAlaAlaGlyGluGlyValThrLeuGlu                              290295300                                                                     TTTGCTCAGAAGTGGATGCACCCCCAAGTGACACCTACTGATGACTCA960                           PheAlaGlnLysTrpMetHisProGlnValThrProThrAspAspSer                              305310315320                                                                  AACCCTTGGTGGGCAGCTTTTAGCCGGGTCTGCAAGGATATGAACCTC1008                          AsnProTrpTrpAlaAlaPheSerArgValCysLysAspMetAsnLeu                              325330335                                                                     ACTCTGGAGCCTGAGATCATGCCTGCTGCCACTGACAACCGCTATATC1056                          ThrLeuGluProGluIleMetProAlaAlaThrAspAsnArgTyrIle                              340345350                                                                     CGCGCGGTGGGGGTCCCAGCTCTAGGCTTCTCACCCATGAACCGCACA1104                          ArgAlaValGlyValProAlaLeuGlyPheSerProMetAsnArgThr                              355360365                                                                     CCTGTGCTGCTGCACGACCACGATGAACGGCTGCATGAGGCTGTGTTC1152                          ProValLeuLeuHisAspHisAspGluArgLeuHisGluAlaValPhe                              370375380                                                                     CTCCGTGGGGTGGACATATATACACGCCTGCTGCCTGCCCTTGCCAGT1200                          LeuArgGlyValAspIleTyrThrArgLeuLeuProAlaLeuAlaSer                              385390395400                                                                  GTGCCTGCCCTGCCCAGTGACAGC1224                                                  ValProAlaLeuProSerAspSer                                                      405                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 408 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetThrSerLysGlyProGluGluGluHisProSerValThrLeuPhe                              151015                                                                        ArgGlnTyrLeuArgIleArgThrValGlnProLysProAspTyrGly                              202530                                                                        AlaAlaValAlaPhePheGluGluThrAlaArgGlnLeuGlyLeuGly                              354045                                                                        CysGlnLysValGluValAlaProGlyTyrValValThrValLeuThr                              505560                                                                        TrpProGlyThrAsnProThrLeuSerSerIleLeuLeuAsnSerHis                              65707580                                                                      ThrAspValValProValPheLysGluHisTrpSerHisAspProPhe                              859095                                                                        GluAlaPheLysAspSerGluGlyTyrIleTyrAlaArgGlyAlaGln                              100105110                                                                     AspMetLysCysValSerIleGlnTyrLeuGluAlaValArgArgLeu                              115120125                                                                     LysValGluGlyHisArgPheProArgThrIleHisMetThrPheVal                              130135140                                                                     ProAspGluGluValGlyGlyHisGlnGlyMetGluLeuPheValGln                              145150155160                                                                  ArgProGluPheHisAlaLeuArgAlaGlyPheAlaLeuAspGluGly                              165170175                                                                     IleAlaAsnProThrAspAlaPheThrValPheTyrSerGluArgSer                              180185190                                                                     ProTrpTrpValArgValThrSerThrGlyArgProGlyHisAlaSer                              195200205                                                                     ArgPheMetGluAspThrAlaAlaGluLysLeuHisLysValValAsn                              210215220                                                                     SerIleLeuAlaPheArgGluLysGluTrpGlnArgLeuGlnSerAsn                              225230235240                                                                  ProHisLeuLysGluGlySerValThrSerValAsnLeuThrLysLeu                              245250255                                                                     GluGlyGlyValAlaTyrAsnValIleProAlaThrMetSerAlaSer                              260265270                                                                     PheAspPheArgValAlaProAspValAspPheLysAlaPheGluGlu                              275280285                                                                     GlnLeuGlnSerTrpCysGlnAlaAlaGlyGluGlyValThrLeuGlu                              290295300                                                                     PheAlaGlnLysTrpMetHisProGlnValThrProThrAspAspSer                              305310315320                                                                  AsnProTrpTrpAlaAlaPheSerArgValCysLysAspMetAsnLeu                              325330335                                                                     ThrLeuGluProGluIleMetProAlaAlaThrAspAsnArgTyrIle                              340345350                                                                     ArgAlaValGlyValProAlaLeuGlyPheSerProMetAsnArgThr                              355360365                                                                     ProValLeuLeuHisAspHisAspGluArgLeuHisGluAlaValPhe                              370375380                                                                     LeuArgGlyValAspIleTyrThrArgLeuLeuProAlaLeuAlaSer                              385390395400                                                                  ValProAlaLeuProSerAspSer                                                      405                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GluTrpGlnArgLeuGlnSerAsnProHisLeuLys                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       LeuGluGlyGlyValAlaTyrAsnValIlePro                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GluGlyTyrIleTyrAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GAGGGGTATATATATGC17                                                           (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AsnProHisLeuLys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TTGAGGTGGGGGTT14                                                              (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      TTTAAGTGGGGGTT14                                                              __________________________________________________________________________

We claim:
 1. A process for preparing protein which comprises:(a)culturing a cell capable of producing a protein in a nutrient culturemedium to form said protein said cell being selected from the groupconsisting of natural human cells, transformed animal cells, andtransformed microorganisms; (b) purifying said protein by combining atleast two techniques from the group consisting of salting out, dialysis,centrifugation, gel filtration, chromatography, ion-exchangechromatography, affinity chromatography, electrophoresis, isoelectricfocusing, and isoelectric fractionation to purify said protein, whereinsaid purified protein has the following physicochemical properties:(1)Molecular weight 45,000±5,000; (2) Isoelectric point pI=5.7±0.5; (3)Partial amino acid sequence Possessing a partial amino acid sequence ofAsp-Ser-Glu-Gly-Tyr-Ile-Tyr-Ala-Arg-Gly-Ala-Gln-Asp-Met-Lys-(SEQ IDNO: 1) or Glu-His-Trp-Ser-His-Asp-Pro-Phe-Glu-(SEQ ID NO: 2); (4)Solubility in solvent Soluble in water, physiological saline andphosphate buffer; (5) Biological activity Exhibitingmetastasis-inhibitory activity on RPMI 4788 cell (FERM BP-2429), anestablished cell line derived from human colon cancer; (6) StabilityInactivated in water at pH 7.2 and 100° C. for 30 minutes and beingstable in water at pH 7.2 and 4° C. for one month; (7) Acute toxicityHaving an LD₅₀ of at least 50 mg/kg mouse on an intravenousadministration; and (c) recovering said protein in purified form.
 2. Theprocess of claim 1, wherein said protein has an amino acid sequenceselected from the group consisting of the amino acid sequence ofchemical formula 1: (SEQ ID No: 4):

    ______________________________________                                        Chemical formula 1                                                            ______________________________________                                        1      Met--Thr--Ser--Lys--Gly--Pro--Glu--Glu--Glu--His--                     11     Pro--Ser--Val--Thr--Leu--Phe--Arg--Gln--Tyr--Leu--                     21     Arg--Ile--Arg--Thr--Val--Gln--Pro--Lys--Pro--Asp--                     31     Tyr--Gly--Ala--Ala--Val--Ala--Phe--Phe--Glu--Glu--                     41     Thr--Ala--Arg--Gln--Leu--Gly--Leu--Gly--Cys--Gln--                     51     Lys--Val--Glu--Val--Ala--Pro--Gly--Tyr--Val--Val--                     61     Thr--Val--Leu--Thr--Trp--Pro--Gly--Thr--Asn--Pro--                     71     Thr--Leu--Ser--Ser--Ile--Leu--Leu--Asn--Ser--His--                     81     Thr--Asp--Val--Val--Pro--Val--Phe--Lys--Glu--His--                     91     Trp--Ser--His--Asp--Pro--Phe--Glu--Ala--Phe--Lys--                     101    Asp--Ser--Glu--Gly--Tyr--Ile--Tyr--Ala--Arg--Gly--                     111    Ala--Gln--Asp--Met--Lys--Cys--Val--Ser--Ile--Gln--                     121    Tyr--Leu--Glu--Ala--Val--Arg--Arg--Leu--Lys--Val--                     131    Glu--Gly--His--Arg--Phe--Pro--Arg--Thr--Ile--His--                     141    Met--Thr--Phe--Val--Pro--Asp--Glu--Glu--Val--Gly--                     151    Gly--His--Gln--Gly--Met--Glu--Leu--Phe--Val--Gln--                     161    Arg--Pro--Glu--Phe--His--Ala--Leu--Arg--Ala--Gly--                     171    Phe--Ala--Leu--Asp--Glu--Gly--Ile--Ala--Asn--Pro--                     181    Thr--Asp--Ala--Phe--Thr--Val--Phe--Tyr--Ser--Glu--                     191    Arg--Ser--Pro--Trp--Trp--Val--Arg--Val--Thr--Ser--                     201    Thr--Gly--Arg--Pro--Gly--His--Ala--Ser--Arg--Phe--                     211    Met--Glu--Asp--Thr--Ala--Ala--Glu--Lys--Leu--His--                     221    Lys--Val--Val--Asn--Ser--Ile--Leu--Ala--Phe--Arg--                     231    Glu--Lys--Glu--Trp--Gln--Arg--Leu--Gln--Ser--Asn--                     241    Pro--His--Leu--Lys--Glu--Gly--Ser--Val--Thr--Ser--                     251    Val--Asn--Leu--Thr--Lys--Leu--Glu--Gly--Gly--Val--                     261    Ala--Tyr--Asn--Val--Ile--Pro--Ala--Thr--Met--Ser--                     271    Ala--Ser--Phe--Asp--Phe--Arg--Val--Ala--Pro--Asp--                     281    Val--Asp--Phe--Lys--Ala--Phe--Glu--Glu--Gln--Leu--                     291    Gln--Ser--Trp--Cys--Gln--Ala--Ala--Gly--Glu--Gly--                     301    Val--Thr--Leu--Glu--Phe--Ala--Gln--Lys--Trp--Met--                     311    His--Pro--Gln--Val--Thr--Pro--Thr--Asp--Asp--Ser--                     321    Asn--Pro--Trp--Trp--Ala--Ala--Phe--Ser--Arg--Val--                     331    Cys--Lys--Asp--Met--Asn--Leu--Thr--Leu--Glu--Pro--                     341    Glu--Ile--Met--Pro--Ala--Ala--Thr--Asp--Asn--Arg--                     351    Tyr--Ile--Arg--Ala--Val--Gly--Val--Pro--Ala--Leu--                     361    Gly--Phe--Ser--Pro--Met--Asn--Arg--Thr--Pro--Val--                     371    Leu--Leu--His--Asp--His--Asp--Glu--Arg--Leu--His--                     381    Glu--Ala--Val--Phe--Leu--Arg--Gly--Val--Asp--Ile--                     391    Tyr--Thr--Arg--Leu--Leu--Pro--Ala--Leu--Ala--Ser--                     401    Val--Pro--Ala--Leu--Pro--Ser--Asp--Ser.                                ______________________________________                                    


3. The process of claim 1, wherein said human cell is derived from humanT-cell leukemia.
 4. The process of claim 3, wherein said human cellderived from human T-cell leukemia is HPB-MLT cell (FERM BP-2430) orMOLT-4 cell (ATCC CRL 1582).
 5. The process of claim 1, wherein the step(a) includes contacting said cell with Bacille Calmette-Guerin andlysaccharide as an inducer of said protein.